Our proposed study will test the hypothesis that the theoretical framework first published by Lizzi and co- workers and subsequently expanded by Insana and Lizzi and their co-workers accurately relates spectral-parameter values to mechanical and geometric properties of isotropic, weak scatterers, insonified by moderately focused transducers. The study also will go beyond rigorous validation of the original theory using its original assumptions to include assessment of spectral behavior under scattering conditions that are outside those assumptions, i.e., scattering for non-isotropic and densely-packed scatterers, scattering with strongly-focused transducers, and scattering over a broad range of frequencies. Success in this study will provide a reliable, more-general basis for quantitative determination of scatterer properties in diagnosing disease, monitoring disease progression or response to therapy, and evaluating tissue properties in basic biological and physiological research. The proposed experimental method for validating the theory will utilize isolated, living and fixed, cultured cells and nuclei suspended in a liquid medium in known concentrations and having known sizes. Acquisition of ultrasound echo signals will utilize broadband, focused transducers with center frequencies ranging from 10 MHz to 75 MHz and with f-numbers ranging from 2 to 4 at each center frequency. Because existing theory utilizes spatial autocorrelation functions and form factors related to the spatial distributions of the acoustic impedances of scatterers, we will compute acoustic impedances for each scatterer type from ultrasound propagation velocities and mass densities measured using cells compacted into pellets by centrifugation. We will generate normalized (system-independent) spectra for all experimental scattering and insonification conditions, and will compare computed spectra to theory. We then will extend our analysis to include alternative methods of estimating scatterer properties, including autoregression and wavelet methods, as well as the commonly used Fourier methods. We also will compare alternative methods of normalization, i.e., those based on deterministic reflections from planar targets and those based on stochastic returns from well-defined scattering targets. The proposed study will give greater insight into phenomena related to scattering of ultrasound by tissue, and will provide a firm foundation for improved ultrasonic means of evaluating and imaging tissue based on the properties of its constituent scatterers. The study will be performed collaboratively by a multidisciplinary team of scientists at the Lizzi Center for Biomedical Engineering at Riverside Research Institute and the Department of Ophthalmology at the Weill Medical College of Cornell University. The proposed project will validate fundamental, but currently incompletely validated, theories of scattering of ultrasound in soft tissues. To date, support for existing scattering theories only has been inferential. Our proposed direct validation approach will lead to refinements of scattering theory that will provide a basis for formulating a more-exact theory and will improve our insights into the way differences among tissues are manifested in their scattering behavior, e.g., differences between normal and diseased or damaged tissue. [unreadable] [unreadable] [unreadable]